1. Field of the Invention
The invention relates to a method for preparing viral suspensions. The invention relates in particular to a method for preparing high-titer viral suspensions in cell cultures. Preferred methods include increasing the volume of the cell culture prior to infection with viral material, and subsequent further steps of expanding the volume to a final volume which is distinctly larger than the maximum culture volume prior to infection.
2. Related Prior Art
The prior art has disclosed various methods for preparing viral material, in particular methods in which the viral material is prepared from animal cell cultures.
The skilled person distinguishes adherently growing cell lines, i.e. cell lines which preferably grow on solid surfaces, from cell lines which preferably grow in suspension. Adherently growing cell lines are either cultured directly on the surface of the culturing vessel used or they grow on solid particles (e.g. on microcarriers) which for their part may be suspended in a nutrient medium.
Methods for preparing viral material are known that use either cell lines growing in suspension or adherently growing cell lines.
Media composition is of great importance in the preparation of viral suspensions using cell cultures. In many cases, fetal calf serum (FCS) and growth factors of animal or plant origin must be added. Besides batch fluctuations and interfering protein components during downstream processing, the use of sera constitutes a biological safety risk (BSE/TSE, mycoplasma, prions etc.). Preference should therefore be given to serum-free, if possible synthetic, media [MERTEN ET AL. 1994].
The use of adherent cells, for example in microcarrier cultures, in particular causes, in addition to the typical technical barriers of scaling up, such as, for example, maintaining a sufficient oxygen supply, removing CO2, adequate homogenization of the fermenter culture with minimum shearing stress, also and in particular problems with the inoculation of the next larger process scale [GLACKEN ET AL. 1983, J. B. GRIFFITHS ET AL. 1985, AMERSHAM 2001].
In this context, “direct migration” of purely adherently growing cells from carrier to carrier can take place only by manipulating the process in such a way that the cells lose at least partially their adherence due to said manipulation. Strategies for removing the adherence of adherently growing cells and enzymes which may be used for this are known to the skilled person [E. LINDNER ET AL. 1987, AMERSHAM 2001, DÜRRSCHMID ET AL. 2003] and must be taken into account in the development of said process with regard to removing or inactivating the enzymes used. In the 1970s and 1980s, successful experiments on direct cell migration from the container surfaces to microcarriers in roller bottles, Petri dishes and T flasks were carried out on smaller scales. A successful migration from carrier to carrier of adherently growing cells is known to the skilled worker only in fixed bed reactors, but this statement must be qualified by the fact that these are cell lines which grow both in suspension and adherently [AMERSHAM 2001, DÜRRSCHMID ET AL. 2003].
Particularly important is the way in which the process is carried out. The literature describes various methods such as, for example, batch or perfusion cultures. Perfusion cultures are used here for decoupling the dwell time from the specific growth rate, for avoiding inhibitions or limitations from the culture medium to increase productivity and are frequently run in “high density cell culture” (HDCC) mode over several months. However, in addition to complicated peripheral equipment (separator, spin filter, ultrasound cell retention, etc.), these systems require lengthy and complex start-up periods [M. REITER ET AL. 1990, GLACKEN ET AL. 1983, J. B. GRIFFITHS ET AL. 1985, AMERSHAM 2001, DÜRRSCHMID ET AL. 2003A].
It is also possible to supply enough nutrients by feeding the cell culture with highly concentrated substrate solutions. Inhibitions resulting from feeding, for example ammonium and/or lactate, may cause lower yields and productivities, in particular in HDCC mode. Up to now perfusion or dialysis systems have recommended themselves for avoiding inhibitory concentrations.
Process control problems may occur in the preparation of viral material by means of animal cell culture, which involves observing complex coupled kinetics of the cells and the virus, in particular when microcarrier cell cultures are used.
For example, the usefulness of propagating a CPE (cytopathic effect)-causing virus by complex perfusion is questionable, since said viruses usually destroy or lyse the cells within short periods of time (sometimes less than 3 to 7 days after infection).
The literature describes batch processes for virus propagation on the pilot and production scales (50 to 1000 l). Virus propagation is carried out with relatively low cell densities in all of the microcarrier processes described. After infection with the virus to be propagated, said infection continues up to the harvest, for example in the later final volume of the production scale [B. MONTAGNON ET AL. 1984, B. BAIJOT ET AL. 1987]. In some cases, perfusion cultures on the laboratory scale for slowly or non-lysing viruses have been described. In one case, a change of media to the original volume is described [AMERSHAM 2001].
U.S. Pat. No. 6,455,298 B1 and U.S. Pat. No. 6,656,720 B2 describe a method for preparing influenza virus material using cell lines growing in suspension. The disclosed method includes a first culturing phase in which the cell material is propagated in suspension culture, an infection step, and subsequently a second culturing phase in which the virus is produced. During this phase, the culture may be diluted further by adding medium or may be run like a perfusion culture. The advantage of this method is the fact that the capacity of said method is not limited by the limited size of the inner surface of the culturing vessels. Disadvantageously, however, a suspension culture cannot achieve cell densities as high as those possible by using microcarrier-based methods for virus production. Furthermore, the removal of cell material from the nutrient medium is considerably more complicated in suspension cultures than in microcarrier-based methods. These disadvantages are avoided in methods according to the present invention, since these make use of adherently growing cell lines on microcarriers for preparing viral material.
U.S. Pat. No. 6,726,907 and WO 95/24468 describe methods for preparing viral material, comprising a first culturing phase for propagating the cell material, an infection step and a subsequent second culturing phase in which the viral material is produced. In contrast to the methods of the invention, no further medium is added during the second culturing phase, and therefore the culture volume is not increased further during said second culturing phase. This results in a relatively small volume harvested, and the culture moreover also has a lower virus titer in comparison with the method of the invention.
U.S. Pat. Nos. 5,994,134, 5,719,051 and 6,194,210 disclose microcarrier-based methods for preparing viral material, which likewise include a first culturing phase, an infection step and a second culturing phase. In contrast to methods according to the present invention, this second culturing phase is not accompanied by any increase in the culture volume but is carried out as a perfusion culture. A continuous flow of fresh medium is supplied, while an equal volume flow of culture medium is removed, and the culture volume therefore remains constant. This method has an advantage over the method described above using suspended cell lines in that firstly a greater cell density can be achieved and secondly large amounts of virus-containing culture medium can be harvested over a longer period of time. However this microcarrier-based method for preparing viral material has a disadvantage in that the virus-containing culture media obtained have a lower virus titer (viral particles per unit volume) compared with the methods of the invention. This makes isolating the viral material more difficult and thereby increases the costs of the product. Furthermore, the supply of fresh medium and simultaneous removal of virus-containing culture broth make great demands on sterilization techniques and increase the risk of contaminations. It is not possible to use methods for preparing viral material with a second culturing phase in perfusion mode, if the virus to be produced causes the lysis of the producing cell and thereby a cytopathic effect (CPE).
This also applies to the complex method of external or internal dialysis, with mass transfer via semipermeable membranes having a specific molecular mass cut-off. To this end, the exhausted medium must be separated from the cells, before it is dialyzed with fresh medium via an externally applied membrane in a countercurrent or cocurrent process. Problems include, aside from the blocking of the membrane within the module by cell debris, etc., especially the complicated apparatus and scaling up.